Browsing by Subject "LSD1"

Lysine specific demethylase 1 (LSD1) is a FAD-dependent amine oxidase enzyme responsible for removing methyl groups from the side chain nitrogen of lysine within histones in order to regulate gene transcription. By its interaction with various transcriptional complexes, including those containing estrogen receptor α (ERα), LSD1 mediates expression of many genes important in cancer proliferation and progression. Herein, we report our efforts towards understanding the function of LSD1 in breast cancer. We have developed a straightforward method for the syntheses of 2-arylcyclopropylamines as irreversible mechanism-based inactivators of LSD1. We employed these small molecules as probes of LSD1 activity, and together with experiments involving the knockout of LSD1 by small interfering RNA (siRNA), we have shown that LSD1 activity is essential for both ERα-postive and ERα-negative breast cancer proliferation. LSD1 inhibitors induce a dramatic cell cycle arrest without causing apoptosis.

Furthermore, we observe that LSD1 and ERα work cooperatively to express certain estrogen-target genes through simultaneous recruitment to promoters; LSD1 inhibition diminishes ERα recruitment. Similarly, knockdown of CoREST, a binding partner of LSD1, results in comparable changes in gene expression. Although, we have not observed a direct interaction between LSD1 and ERα, we believe that CoREST may be facilitating this interaction. We have made efforts to inhibit the interaction between LSD1 and CoREST in vitro in hopes of targeting this interface in breast cancer cells in order to disrupt the necessary functional complex and prevent LSD1 activity.

The post translational modification (PTM) of histone proteins is a highly dynamic process that is utilized in the control of gene transcription. This epigenetic process involves enzymatic ‘writers’ and ‘erasers’ which place or remove chemical modifications to the unstructured tails of histone proteins which protrude out from the nucleosomal core. In a highly dynamic manner, each PTM is spatiotemporally regulated and combinations of PTMs at a gene promotor or enhancer region leads to transcriptional enhancement or repression. The gene targets as well as selectivity and specificity of epigenetic enzymes is regulated by the multimeric complexes each enzyme is co-opted. Each complex contains a unique set of coregulatory proteins with RNA and DNA binding domains and PTM ‘reader’ domains to direct the catalytic machinery to a specific subset of genes. The coregulatory proteins also affect the specificity and selectivity of the enzyme through mechanisms which are only beginning to be explored.

Our interest is in elucidating the role of coregulatory proteins and lncRNA with respect to lysine-specific demethylase 1 (LSD1/KDM1A). A flavin-dependent mono-and di-demethylase of H3K4me1/2 and H3K9me1/2, KDM1A has been implicated in many different multimeric enzymatic complexes which, in some cases such as the REST and NuRD complexes, function on opposing pathways. This disparity in the downstream outcome being coordinated by the same enzyme highlights the need to understand not only epigenetic enzymes, but to consider the complexes as a whole towards therapeutic targeting.

The specific aims of my thesis were to (a) interrogate the role of individual and multiple coregulatory partners in enzyme selectivity and specificity (b) establish tools to study the mechanisms of biochemical and biophysical of protein:protein and protein:lncRNA interactions and (c) elucidate key characteristics of protein:protein and protein:lncRNA interfaces towards targeted disruption. To this end, I have utilized cloning and mutagenesis methods to heterologously express and purify coregulatory partners of KDM1A in E. coli. I chose coregulatory partners found in a common catalytic core as well as several additional coregulatory proteins from a stable KDM1A-containing 5-mer complex. I have produced multiple constructs for four of these proteins to allow for multiple affinity purification routes as well as for future binding studies. I have further expressed each of these constructs and have made significant efforts towards the purification of each construct based on solubility.

I furthermore established HDX-MS and SELEX protocol in our lab as tools to allow us to explore the dynamics of these epigenetic interactions. I further demonstrated and confirmed that there is no hotspot along the binding interface between KDM1A and CoREST, but that CoREST stabilizes the apical end of the KDM1A tower domain via HDX-MS with the highest change in deuterium uptake, over 20%, long KDM1A TαA residues 440-451.

I also made significant efforts towards elucidating the interaction between KDM1A and HOTAIR. Firstly, I established an RNA radiolabeled EMSA assay for the lab which allowed us to test the binding of HOTAIR to KDM1A. With this assay, we saw that CoREST286-482, specifically the linker region (residues 293-380), must be bound to KDM1A for HOTAIR to bind and that the dissociation constant was unchanged at 1.710.38 µM and 1.29±0.34 µM, respectively. Further, I confirmed that the first 320 nt of domain 4 of HOTAIR (nt 1500-1820) contain the critical binding and that the dissociation constant was slightly higher at 2.97±0.96 µM.

I have also optimized SHAPE-MaP and crosslinking strategies to explore the binding interface between KDM1A:CoREST286-482 and lncRNA. I determined that there were 83 nt that displayed at least a 1.5-fold change in SHAPE reactivity of HOTAIR D4 due to the presence of KDM1A:CoREST286-482. I also utilized a free-energy based secondary structure model to establish a secondary structure for HOTAIR D4 based on my SHAPE-MaP data. I noted that 44% of the significant nt were confined to a stretch of RNA (nt 1538-1610, 1779-1844) that is predominantly dsRNA. Further usage of photochemical crosslinking strategies revealed a propensity for G:C paired nt to be crosslinked to KDM1A:CoREST286-482. A similar nt sequence around these paired nt suggests a binding motif. The implications of these results is discussed herein.

Lysine-specific demethylase 1 (LSD1/KDM1A) regulates transcriptional events by post-translational modifications of histone H3 tails at residues K4 an K9. This enzyme plays a vast number of roles in both normal cellular functions and diseases states. Increasingly it is appreciated that this enzyme, like most epigenetic regulators, does not function alone, but rather forms a catalytic subunit of much larger protein assemblies that congregate on chromatin to concertedly mediate transcriptional events. LSD1 in particular has been found in many different complexes, in many different tissues and can facilitate both activation and repression events.

Because of these roles, LSD1 is viewed as a potential therapeutic target. Significant effort has recently led to the development of highly selective and potent active-site inhibitors. These inhibitors have particularly shed light on the cancer-promoting activities of LSD1 in acute myeloid leukemia and small cell lung carcinoma. However, one failing of these strategies is that active site inhibition is incapable of differentiating between the multitude of functions LSD1 performs. We sought to address this issue by instead developing first-generation tools to explore protein-protein interaction disruption as an alternative strategy for inhibiting the enzyme.

To this end, we have carefully examined a well-characterized interaction between LSD1 and the scaffolding protein CoREST. Using this interaction as a template, we developed a probe we show can compete with CoREST for interaction with LSD1. Furthermore, we generated cell permeable versions of this probe and examined the effects in a model of breast cancer. We find that our probe can selectively inhibit estrogen signaling, a feat that was not possible with current small molecule inhibition or RNA interference technologies. We therefore we propose that disrupting interactions such as this is an excellent alternative for targeting “undruggable” proteins, but also may also expand the current therapeutic space by granting precise control over the individual functions of proteins.